The response regulator protein, NarL, is a member of a regulatory circuit involving dual two-component signal transduction systems that mediate nitrate regulation of anaerobic respiratory gene expression in E. coli. This research proposal analyzes the regulatory characteristics of the N-terminal and C-terminal domains of the NarL response regulatory protein to determine their roles in DNA binding specificity, affinity and transcriptional activation/repression. The issues that will be addressed include whether DNA binding by NarL is cooperative; whether the DNA-binding specificity of NarL resides in the C- terminal domain of the protein, and if so, are the transcriptional regulatory properties of NarL also located in the C-terminus; whether the N-terminal domain plays a role in the DNA binding affinity of NarL; and finally, what is the Kon and Koff of NarLWT, NarLWT-Phosphate and the C-terminal domain of NarL for various NarL-regulated target sequences and does mutational analyses of the regulatory sequences alter the rate constants. The use of both in vitro (DNase I footprint analysis, filter binding assays, gel shift analysis, site-directed mutagenesis, etc.) and in vivo expression studies will be employed to investigate these questions. The information gained from the proposed research will not only broaden our knowledge of the NarXQLP nitrate-regulatory systems, but will also enhance our knowledge of the global family of two component regulatory systems involved in many aspects of bacterial growth and survival.